Cold induced expression of a novel levansucrase gene sacB1 enhances exopolysaccharide production and stress resilience in Leuconostoc mesenteroides

Abstract

Exopolysaccharides (EPS) play critical roles in microbial survival, stress adaptation, and biofilm formation across diverse environments. In food-associated bacteria such as Leuconostoc mesenteroides, understanding the regulation of EPS production under environmental stress is important for both spoilage control and industrial applications. However, the mechanisms linking cold stress to EPS biosynthesis remain poorly understood. Here, we show that sucrose and low temperature (8 °C) trigger a metabolic shift from dextran-only to combined dextran and levan biosynthesis in four meat-borne Leuc. mesenteroides strains. Two high EPS-producing strains (HEPRs) possess the sacB_1 gene, which encodes a previously uncharacterized levansucrase absent from low EPS-producing strains (LEPRs) that only carry the levS gene. This is the first study to describe the role of sacB_1 in cold-induced EPS production. Notably, sacB_1 was also identified in Leuc. mesenteroides strains isolated from plant-based fermentations such as kimchi and birch sap, but the HEPR strains analyzed here are the only known meat-derived isolates to carry this gene. Genomic analyses revealed highly conserved biosynthetic clusters for dextran, heteropolysaccharide, and levan. Gene expression profiling showed that levS and sacB_1 were upregulated at 8 °C, while dsrD expression was favoured at 25 °C. Cold-induced sucrose metabolism, characterized by high expression of levS, sacB_1, and dsrD, enhanced cell viability under oxidative stress. Furthermore, heterologous expression of sacB_1 in Leuc. mesenteroides and Lactococcus lactis improved resilience under cold and high-aeration conditions, confirming the protective role of levan. These findings advance the understanding of temperature-dependent EPS regulation in LAB and highlight sucrase diversity as a key factor in microbial adaptation to environmental stress.

Keywords: Leuconostoc mesenteroides; Cold stress; Exopolysaccharide; Levansucrase; Sucrose-induced metabolism.

Fig. 1

Effect of carbon sources and temperature on EPS production. a Heatmap representing the qualitative effect of temperature (8 °C, 25 °C, 37 °C) and carbon source (glucose, fructose, sucrose) on EPS production by four Leuc. mesenteroides strains (KS273, KS279, KS276, KS277). Higher EPS production is indicated by red/yellow coloration; b Visual comparison of high EPS producers (HEPRs, KS273, KS279) and low EPS producers (LEPRs, KS276, KS277) in round-bottom well microtiter plates after 5 days of incubation in 5% sucrose at 8 ºC; c Response Surface Methodology (RSM) 3D plots (left) and 2D contour plots (right) showing the quantitative effect of temperature and sugar (sucrose or glucose) concentrations on EPS production for Leuc. mesenteroides strains KS273 (top) and KS276 (bottom)

Fig. 2

Genomic analysis of EPS biosynthetic clusters. a Heatmap of frequency of carbohydrate-active enzymes (CAZymes) from Leuc. mesenteroides high EPS producer strains (HEPRs: KS273, KS279) and low EPS producers (LEPRs: KS276, KS277); b Gene cluster organization of the proposed HePS-Levan (HLBC) and glucan (GBC) biosynthetic gene clusters in HEPRs and LEPRs. The gene clusters were visualized using Clinker on the CAGECAT platform, highlighting the levansucrase genes (levS and sacB_1) and glucansucrase genes (dsrD, gtfN and gtfO); c Annotation and presence/absence comparison of key proteins of the HLBC involved in HePS and levan biosynthesis pathways across the four Leuc. mesenteroides strains. Genes in the HePS operon were categorized into colour groups based on the putative or established functions of their products as in Zeidan et al. (2017); Poulsen, Derkx and Oregaard (2019)^,: Modulation (yellow; phosphoregulatory module epsBCD), polysaccharide assembly (green; initiation epsE, polymerization wzy, export/flippase wzx and attachment epsA/lytR), GT (orange; glucosyltransferases) which assemble the repeating units, and non-housekeeping functions (pink) required for the synthesis of activated sugar precursors and modification of the sugar residues; d Conserved domains (CDs) of levS and sacB_1 from the NCBI Conserved Domain Database

Fig. 3

Gene expression and viability analysis of Leuc. mesenteroides KS273 and KS276 under different carbon sources and temperatures. a Heatmap of gene expression (Log2 TPM) of Wzy-dependent HePS biosynthesis pathway in KS273 and KS276; b Expression of sucrases genes (glucansucrases dsrD, gtfN and gtfO and levansucrases levS, sacB_1) in KS273 (top) and KS276 (bottom) cells grown in M17 with 1.5% glucose (G), sucrose (S) or no sugar (NS) at 25 or 8 ºC for 72 h, represented as transcripts per million (TPM); c ratio of KS273 and KS276 GTs TPM in each condition; d GT gene enrichment expressed as % GT transcripts in KS273 (left) and KS276 (right) at 25 ºC and 8 ºC in M17 with 1.5% sucrose; e Growth curve, f Final cell viability and g zymogram of GTs from KS273 (left) and KS276 (right) cultures grown in M17 with 1.5% glucose (Glu), sucrose (Suc), fructose (Fru) or no sugar (NS) at 8 ºC for 120 h. Zymogram displaying polymers synthesized in situ by DsrD (173 kDa), LevS (111 kDa) and SacB_1 (115 kDa) revealed by Periodic acid-Schiff (PAS) gel staining and ethanol polysaccharide precipitation. The latter is a composite image from two parts of the gel separated by the Commassie Blue-stained Protein Standard (10–200 kDa, New England Biolabs). Full-length gels are displayed in Supplementary Fig. S2. Data are presented as mean ± SD, n = 3. For growth curves and survival plot, two-way repeated measures ANOVA and Tukey’s post-hoc test was carried out: ****p < 0.0001, ***p < 0.001, **p < 0.01, *p < 0.05

Fig. 4

Protective effect of sucrose-induced metabolism (SIM) and levan. a Viability of Leuc. mesenteroides KS273 and KS276 after growing in different levels of oxidative stress: high aeration, (high surface: volume ratio, HA) or low aeration (low surface: volume ratio, LA) in the presence of glucose (Glu) or sucrose (Suc); b growth trend and c final viability of KS276-E (sacB_1⁻) and KS276-SB1 (sacB_1⁺) at 8 ºC under LA in presence of Glu or Suc. d Cell pellets of KS276 (WT) and KS276-SB1 (sacB_1⁺) after growth (120 h) in the presence of Suc under HA in falcon tubes (top) or round-bottom 96-well plate (bottom). e Viability of L. lactis MG1363 (sacB_1⁻) and MG1363-SB1 (sacB_1⁺) after growing for 24 h at 25 ºC in HA or LA in the presence of Suc. f) L. lactis MG1363 (WT) and MG1363-SB1 (sacB_1⁺) colonies after growing for 48 h at 25 ºC on M17 agar plates supplemented with 1.5% sucrose. Data are presented as mean ± SD, n = 3. Kruskal–Wallis test and Dunn’s post-hoc test: ***p < 0.001, **p < 0.01, *p < 0.05